CN102242183B - Morel strain identifying method - Google Patents
Morel strain identifying method Download PDFInfo
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- CN102242183B CN102242183B CN2011101624901A CN201110162490A CN102242183B CN 102242183 B CN102242183 B CN 102242183B CN 2011101624901 A CN2011101624901 A CN 2011101624901A CN 201110162490 A CN201110162490 A CN 201110162490A CN 102242183 B CN102242183 B CN 102242183B
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Abstract
The invention belongs to the technical field of microbes, and relates to a morel strain identifying method. The method comprises the following steps of: firstly, observing the mycelia of the strain to be identified on the surface of a culture medium, if the observed mycelia on the surface of the culture medium have the typical morphological characteristics of morel mycelia on the surface of the culture medium, primarily judging that the strain is the morel strain; and further, observing the substrate mycelia, if the observed substrate mycelia have the morphological characteristics of the morel substrate mycelia, determining that the strain is the morel strain. The morel strain identifying method provided by the invention is easy in operation and low in cost, and does not need special instruments and equipment, and the identifying result is easy to judge and reliable.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of authentication method of morel bacterial classification.
Background technology
Morel is the general designation of morchella (Morchella) fungi, the whole world has reported that more than 50 plant, China has reported that more than 10 plant (about defining of morchella species, difference is larger between the different researchers, some scholar only admits 3～6 species titles of morchella), they all are the rare economic epiphytes with important edible and pharmaceutical use.
The morel bacterial classification refers to move in the Morchella esculenta (L.) Pers mycelium on certain substratum, is important Biological resources, also is the important materials in related scientific research, the teaching.
Utilization to morel mainly contains two kinds of approach.The first is utilized the sporophore of morel, Morchella esculenta (L.) Pers sporophore in the market is mainly derived from field acquisition, and rare because of the source of goods, price is always very high, home market price generally is stabilized in 400～500 yuan of per kilograms, and the world market valency is up to per kilogram 200 dollars.Utilize the morel bacterial classification to produce its sporophore by tame method, it is one of mycologist and edible fungus culturing person problem of doing one's utmost to explore for over 100 years more than a hundred years, and, the report of producing Morchella esculenta (L.) Pers sporophore by artificial culture is all arranged both at home and abroad, but the commercialization artificial culture of unrealized morel so far.The morel artificial cultivation technique of having reported all rests on the accidental generation of sporophore and the very low level of output, also has sizable gap apart from commercialization production.At present, in many places, still underway about the tame exploration of morel.
Another approach to the morel development and use is to utilize the morel bacterial classification to produce the mycelium product by the method for industrial fermentation.Commercially produced Morchella esculenta (L.) Pers mycelium as seasonings since the sixties in last century abroad.
No matter be the scientific research of being engaged in the aspects such as morel artificial culture exploration, still carry out the industrial fermentation production of morel, all need to utilize the morel bacterial classification.Whether the morel bacterial classification, differentiating it really is the morel bacterial classification if being carried out authenticity, be an important process of related scientific research, production and culture presevation unit.
Morel is a kind of fungi that can produce large-scale sporophore, belongs to the macro fungi category, and about the strain identification of macro fungi, traditional method is to make it produce sporophore by artificial culture, then identifies according to the feature of sporophore.Because morel does not produce sporophore substantially under tame condition, just very individual other unit turned out Morchella esculenta (L.) Pers sporophore in very accidental situation, and this is so that the method for the morel bacterial classification being identified according to the sporophore feature is difficult to realization.Study for being engaged in morel, if the bacterial classification of using is false, its result of study just can not be correct, and this also is that morel is studied one of major reason that does not make great progress for a long time.For the unit that is engaged in the Morchella esculenta (L.) Pers mycelium fermentative production, if the bacterial classification of using is false, also exist the possibility that causes higher threat.
Then the development of Protocols in Molecular Biology carries out the strain identification of the fungies such as morel so that people can utilize the mycelium of fungi to measure its specific dna sequence dna according to its specific dna sequence.But this evaluation based on Protocols in Molecular Biology needs relatively more expensive plant and instrument and professional scientific and technical personnel, and the fund of cost is also more, and many units there is no method to be carried out.
Summary of the invention
The purpose of this invention is to provide a kind of morel Strain identification method based on the mycelium morphology feature, overcome the shortcoming of the above two false morels Strain identification method, be implemented under the simple and easy condition Rapid identification to the morel bacterial classification.
The objective of the invention is to be achieved through the following technical solutions: the media surface mycelia of observing first bacterial classification to be identified, if the media surface mycelia that observes has the representative configuration feature of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification, further observe again its substrate mycelium, if the substrate mycelium that observes has the morphological specificity of morel substrate mycelium, can judge that this bacterial classification is exactly the morel bacterial classification.Concrete steps are as follows:
(1) in a culture dish, puts side by side two slide glasss (staying the gap between two slide glasss), with fusing and be cooled to 45～50 ℃ PDA substratum and be poured on the slide glass, on every slide glass, all make the plate culture medium of the about 2～3mm of thickness; Get the middle part that mycelium is inoculated into respectively every plate culture medium with inoculating needle from the bacterium colony of bacterial classification to be identified, cover the lid of culture dish, be placed in 18～20 ℃ the incubator and cultivate; When the new colony radius that grows at above-mentioned plate culture medium when bacterial classification to be identified reaches 1～1.5cm, namely can be used for the evaluation of following steps;
(2) one in the described culture dish of step (1) slide glass that carries bacterial classification bacterium colony to be identified is taken out, with lens wiping paper (without the one side of substratum) below the slide glass cleaned, distilled water is dripped at the place at colony edge, covered sucks unnecessary water with thieving paper from the cover glass edge;
(3) will put microscopically through the above-mentioned slide glass of processing through step (2) observes, with the morphological specificity of media surface mycelia as the important evidence that determines whether morel, if the media surface mycelia that observes has the representative configuration feature of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification; The representative configuration feature of described morel media surface mycelia is: mycelia has obvious trunk, and longer as the mycelia of trunk, straight and upright, thickness is more even, and barrier film is less, two paraclades; The branch that bears from trunk has two classes, and class branch repeatedly in very short distance wherein is the dendroid of gathering, and it is all shorter that these consist of the sprigs that assemble arborizationses, and thickness is inhomogeneous, and is also not straight; Another kind of branch and trunk plesiomorphism from trunk bears can be described as secondary trunk; Branch characteristic on the secondary trunk is similar to trunk, can produce two class branches, and wherein a class is again the trunk of one-level, another kind of in very short distance branch repeatedly, be the dendroid of gathering; Though can produce secondary trunk on the trunks at different levels, the secondary trunk number that produces is less, most of branches of generation be short and small and in very short distance repeatedly the gathering arborizations of branch (see Fig. 1-Fig. 3);
(4) slide glass that another piece in the described culture dish of step (1) is carried bacterial classification bacterium colony to be identified takes out, cut the substratum fritter that size is about 0.3 * 0.3cm in the place that bacterium colony is arranged, and cut the part (namely cutting away surperficial one deck substratum that bacterium colony is arranged) of the thick about 1mm in this substratum fritter surface with blade;
(5) cut away the thin slice (tangent plane is surperficial parallel with plate culture medium) that surperficial substratum fritter is cut into thick about 1～2mm with step (4) is described, the substratum thin slice that is cut into is placed water droplet on the slide glass, covered, and the pressing cover slide is squeezed into skim with the substratum thin slice, then slide glass is put microscopically and observed, the mycelia that observes namely is substrate mycelium.If the substrate mycelium that observes has the morphological specificity of morel substrate mycelium, can judge that this bacterial classification is exactly the morel bacterial classification.The substrate mycelium of morel is to form to the substratum growth inside by being the top of assembling arborizations on this bacterium culture medium surface mycelia, has following morphological specificity: the mycelia plucked, not straight, color is more shallow than the media surface mycelia look of morel, and branch is many and irregular; As a whole, the mycelia of the substrate mycelium of morel and media surface has difference clearly.
In a word, if through above step, the media surface mycelia and the substrate mycelium that observe have respectively the feature of morel media surface mycelia and substrate mycelium, can judge that this bacterial classification is the morel bacterial classification; If through above step, the media surface mycelia that observes does not have the morphological specificity of morel media surface mycelia, the substrate mycelium that perhaps observes does not have the morphological specificity of morel substrate mycelium, can judge that the bacterial classification of identifying is not the morel bacterial classification.
Advantage of the present invention is: simple to operate, do not need special different instrument and equipment, and cost is low, and qualification result is judged easily, and qualification result is comparatively reliable.
Description of drawings
Fig. 1～3 are the media surface mycelia of morel;
Fig. 4～6 are the media surface mycelia of non-morel.
Embodiment
Morel Strain identification method of the present invention, concrete steps are as follows:
(1) in a culture dish, puts side by side two slide glasss (leaving the gap between two slide glasss), with fusing and be cooled to 45～50 ℃ PDA substratum and be poured on the slide glass, on every slide glass, all make the plate culture medium of the about 2～3mm of thickness; Get the middle part that mycelium is inoculated into respectively every plate culture medium with inoculating needle from the bacterium colony of bacterial classification to be identified; Cover the lid of culture dish, be placed in 18～20 ℃ the incubator and cultivate; When the new colony radius that grows at above-mentioned plate culture medium when bacterial classification to be identified reaches 1～1.5cm, namely can be used for the evaluation of following steps;
(2) with one in the described culture dish of step (1) carry bacterial classification bacterium colony to be identified slide glass take out, with lens wiping paper (without the one side of substratum) below the slide glass cleaned, distilled water is dripped at the place at colony edge, covered sucks unnecessary water with thieving paper from the cover glass edge;
(3) above-mentioned slide glass is put microscopically and is observed, with the morphological specificity of media surface mycelia as the important evidence that determines whether morel; If the media surface mycelia that observes has the morphological specificity (shown in Fig. 1～3) of morel media surface mycelia, can judge tentatively that this bacterial classification is the morel bacterial classification.The morphological specificity of morel media surface mycelia is: mycelia has obvious trunk, and longer as the mycelia of trunk, straight and upright, thickness is more even, and barrier film is less, two paraclades; The branch that bears from trunk has two classes, and class branch repeatedly in very short distance wherein is the dendroid of gathering, these consist of assemble arborizationses sprigs all short, thickness is inhomogeneous, also not straight; Another kind of branch and trunk plesiomorphism from trunk bears can be described as secondary trunk.Branch characteristic on the secondary trunk is similar to trunk, can produce two class branches, and wherein a class is again the trunk of one-level, another kind of in very short distance branch repeatedly, be the dendroid of gathering.Though can produce secondary trunk on the trunks at different levels, the secondary trunk number that produces is less, most of branches of generation be short and small and in very short distance the gathering arborizations of branch repeatedly;
(4) another piece in the described culture dish of step (1) is carried bacterial classification bacterium colony to be identified slide glass take out, cut the substratum fritter that size is about 0.3 * 0.3cm in the place that bacterium colony is arranged, and cut the part (namely cutting away surperficial one deck substratum that bacterium colony is arranged) of the thick about 1mm in this substratum fritter surface with blade;
(5) cut away the thin slice (tangent plane is surperficial parallel with plate culture medium) that surperficial substratum fritter is cut into thick about 1～2mm with above-mentioned, the substratum thin slice that is cut into is placed water droplet on the slide glass, covered, and the pressing cover slide is squeezed into skim with the substratum thin slice, then slide glass is put microscopically and observed, the mycelia that observes namely is substrate mycelium; If the substrate mycelium that observes has the morphological specificity of morel substrate mycelium, can judge that this bacterial classification is exactly the morel bacterial classification.The morphological specificity of morel substrate mycelium is as follows: the mycelia plucked, and not straight, color is more shallow at the mycelia look of media surface than morel, and branch is many and irregular.As a whole, the mycelia of the substrate mycelium of morel and media surface has difference clearly.The substrate mycelium of morel is to be the top of assembling arborizations on the media surface mycelia by morel to form to the substratum growth inside.
If through above step, the media surface mycelia that observes does not have the representative configuration feature of morel media surface mycelia, the substrate mycelium that perhaps observes does not have the morphological specificity of morel substrate mycelium, can judge that the bacterial classification of identifying is not the morel bacterial classification.
Respectively the more or less a hundred bacterial classification material that includes the morel bacterial classification has been carried out qualification test with method of the present invention with based on the molecular assay method (contrast) of rDNA the Internal Transcribed Spacer (ITS) sequence, test confirms that two kinds of qualification results are in full accord.See the following form.
The qualification result contrast of authentication method of the present invention and molecular methods
Claims (1)
1. morel Strain identification method is characterized in that concrete steps are as follows:
(1) in a culture dish, puts side by side two slide glasss, with fusing and be cooled to 45～50 ℃ PDA substratum and be poured on the slide glass, on every slide glass, all make the plate culture medium of thickness 2～3mm; Get the middle part that mycelium is inoculated into respectively every plate culture medium with inoculating needle from the bacterium colony of bacterial classification to be identified; Cover the lid of culture dish, be placed in 18～20 ℃ the incubator and cultivate; When the new colony radius that grows at above-mentioned plate culture medium when bacterial classification to be identified reaches 1～1.5cm, be used for the evaluation of following steps;
(2) one in the described culture dish of step (1) slide glass that carries bacterial classification bacterium colony to be identified is taken out, clean below slide glass with lens wiping paper, distilled water is dripped at the place at colony edge, and covered sucks unnecessary water with thieving paper from the cover glass edge;
(3) the above-mentioned slide glass of processing through step (2) is put the mycelia that microscopically is observed media surface, if the media surface mycelia that observes has the representative configuration feature of morel media surface mycelia: mycelia has obvious trunk, mycelia as trunk is longer, straight and upright, thickness is more even, barrier film is less, two paraclades, the branch that bears from trunk has two classes, class branch repeatedly in very short distance wherein, the dendroid that is gathering, it is all shorter that these consist of the sprig that assembles arborizations, thickness is inhomogeneous, not straight yet, the another kind of branch and the trunk plesiomorphism that bear from trunk, can be described as secondary trunk, branch characteristic on the secondary trunk is similar to trunk, can produce two class branches, wherein a class is again the trunk of one-level, another kind of in very short distance branch repeatedly, be the dendroid of gathering, though can produce secondary trunk on the trunks at different levels, but the secondary trunk number that produces is less, most of branches of generation be short and small and in very short distance the gathering arborizations of branch repeatedly, can judge tentatively that this bacterial classification is the morel bacterial classification;
(4) another piece in the described culture dish of step (1) is carried bacterial classification bacterium colony to be identified slide glass take out, cut the substratum fritter of the about 0.3cm * 0.3cm of size in the place that bacterium colony is arranged, and cut the part of the surperficial thick about 1mm of this substratum fritter;
(5) cut away the thin slice that surperficial substratum fritter is cut into thick 1～2mm with step (4) is described, the assurance tangent plane is surperficial parallel with plate culture medium, the substratum thin slice that is cut into is placed water droplet on the slide glass, covered, and the pressing cover slide is squeezed into skim with the substratum thin slice, then slide glass being put microscopically observes, the mycelia that observes namely is substrate mycelium, if the substrate mycelium that observes has the morphological specificity of morel substrate mycelium: the mycelia plucked, not straight, color is more shallow than the media surface mycelia look of morel, and branch is many and irregular, can judge that this bacterial classification is exactly the morel bacterial classification.
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| CN107177663A (en) * | 2017-06-02 | 2017-09-19 | 武汉市农业科学技术研究院蔬菜科学研究所 | A kind of authentication method of hickory chick bacterial strain aging |
| CN107177514A (en) * | 2017-07-06 | 2017-09-19 | 江油市静远食用菌科技有限责任公司 | A kind of purity is the preparation method of 100% morchella mother culture |
| CN111721747A (en) * | 2020-06-30 | 2020-09-29 | 常熟理工学院 | Method for detecting concentration of morchella hypha nucleuses by cell nucleus staining |
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